Key central metabolites such as ATP, ADP, GTP, GDP, NAD+, NADH and many other small molecules play critical roles in metabolism by modulating the activity of a variety of protein complexes. Metabolic misregulation can result in diseases such as neurodegeneration, cancer and immune disorders. Thousands of enzymes and enzyme isoforms have been biochemically characterized; X-ray crystallographic and NMR spectroscopic analyses have resulted in structural insights into the effects of metabolite binding on protein structure in many instances. To better understand the structural origins of fundamental regulatory mechanisms in allosteric enzymes, we have focused on cryo-EM analysis of glutamate dehydrogenase, a hexameric inner mitochondrial enzyme that is found in all organisms. Glutamate dehydrogenase (GDH) is a highly conserved enzyme expressed in most organisms. GDH plays a central role in glutamate metabolism by catalyzing the reversible oxidative deamination of glutamate to generate -ketoglutarate and ammonia, with the concomitant transfer of a pair of electrons to either NAD+ or NADP+. Regulation of GDH is tightly controlled through multiple allosteric mechanisms. The transition between closed and open states of GDH is modulated by two allosteric sites in each protomer of this enzyme. Although there are numerous crystal structures available for GDH in complex with cofactors and nucleotides, they are limited to the combinations that have been amenable to crystallization. Nearly all X-ray structures of mammalian GDH are in the closed conformation, and the few structures that are in the open conformation are at lower resolution. In our recent work, we have shown using single particle cryo-EM that under several physiologically relevant conditions, GDH enzyme complexes co-exist in both closed and open conformations. We show that the structures in both states can be resolved at near-atomic resolution, with our studies suggesting a molecular mechanism for synergistic inhibition of GDH by NADH and GTP. Our structural studies have established that whether or not GTP is bound, NADH binding is detectable at catalytic and regulatory sites, in both the open and closed conformational states. While the orientation in which NADH binds at the catalytic site is similar for both conformations, the orientation of the nicotinamide portion of NADH in the regulatory site is different between the open and closed conformations. In the closed state, the nicotinamide moiety is inserted into a well-defined cavity at the interface between two adjacent protomers in the trimer. As mentioned above, this cavity is much narrower in the open state, suggesting that this cavity may be unavailable to the NADH nicotinamide moiety when the enzyme is in the open conformation. These structural features provide a potential explanation of the weaker density for the nicotinamide moiety of NADH in the open state, and may account for the higher reported affinity of NADH for the closed state. The role of the nicotinamide moiety in acting as a wedge that prevents the transition to the open conformation also suggests a structural explanation of the mechanism by which NADH binding inhibits the activity of the enzyme by stabilizing the closed conformation state. The rapid emergence of cryo-EM as a tool for near-atomic resolution structure determination provides new opportunities for complementing atomic resolution information from X-ray crystallography, as illustrated here with GDH. Perhaps the most important contribution of these methods is the prospect that when there are discrete sub-populations present, the structure of each state can be determined at near-atomic resolution. What we demonstrate here with GDH is that by employing 3D image classification approaches, we can not only isolate distinct, co-existing conformations, but we can also localize small molecule ligands in each of these conformations. These kinds of approaches are likely to become increasinglyimportant in molecular pharmacology, especially in the context of better understanding drug-target interactions in dynamic protein complexes. These studies were published in the journal Molecular Pharmacology and featured on the cover of the June 2016 issue of the journal.